Some heterologous polypeptides, including bovine somatotropin (BSt), are difficult to express in most E. coli expression systems. Modifications within the first 4 codons of the CDNA encoding the BSt structural gene result in increased levels of BSt expression when these modified cDNAs are expressed in a pBR322-related vector. These modified cDNAs are expressed at even higher levels when placed in a runaway expression vector (U.S. patent application Ser. No. 016,294, filed 19 Feb. 1987 and incorporated herein by reference). The modified ribosome binding sites of the instant invention produce expression of heterologous polypeptides with a non-runaway vector, for example, a derivative of plYR322, equivalent to that achieved with a runaway vector.
Generally, methods for cloning and expressing heterologous polypeptides in transformed hosts are well known to those skilled in the art. Such heterologous polypeptides include, for example, human insulin, growth hormone, interferon and factor VIII, viral antigens, and other animal hormones.
The ribosome binding site is one of the elements involved in such expression. It covers the region about 20 nucleotides on both sides of the initiation codon and contains the Shine-Dalgarno sequence usually 6-10 nucleotides upstream from the initiation codon. The 20 nucleotides after the initiation codon includes the beginning of the gene sequence inserted for expression and often cannot be subjected to modifications without changing the amino acid sequence of the gene. Therefore, in a practical sense, manipulation of the ribosome binding site can only be carried out at the region upstream from the initiation codon. The ribosome binding site is known to function best if it contains certain bases but there is no known requirement for any particular base in a particular sits except that the Shine-Dalgarno sequence is generally purine-rich. The subtle differences among various known ribosome binding site sequences do not provide an obvious comparison to predict which genes will be highly and which will be poorly expressed when associated therewith. A preferred embodiment of the present invention utilizes sequences that are rich in A and T nucleotides to flank the Shine-Dalgarno sequence thereby producing ribosome binding sites that have an increased number of adenine and thymidine residues as compared to the naturally occurring ribosome binding sites from which they are derived.
Naturally occurring BSt is a mixture of heterogeneous proteins, the amino acid sequences of which are known (Paladini, A. C., et al., Molecular Biology of Growth Hormone, CRC Reviews in Biochem., 15(1):25-56 (1983). The naturally occurring mixtures have been purified from pituitary glands of cattle. The commercial potential for using BSt for promoting growth and lactation is well recognized and documented by biological studies on both dairy and feed cattle (Eppard, P. J. and Bauman, D. E., The Effect of Long-Term Administration of Growth Hormone on Performance of Lactating Dairy Cows; and Bausan, D. E., Effect of Growth Hormone on Growth Rates and Mammary Development of Ruminants, Proc. 1984 Cornell Nutrition Conference for Feed Manufacturers, pp. 5-17, published by Cornell University, Ithaca, N.Y.).
Recombinant bovine somatotropin (rBSt) can be produced in transformed microorganisms using a variety of recombinant genetic plasmids (see, e.g., Seeburg, P. H., et al., "Efficient Bacterial Expression of Bovine and Porcine Growth Hormones," DNA, 2:37-45 (1983)); European Patent Application 47 600; United Kingdom Patent Application, GB 2073245A; Schoner, B. E., et al., Role of mRNA Translational Efficiency in Bovine Growth Hormone Expression in Escherichia coli, PNAS USA, 81:5403-5407 (1984); European Patent Application 103 395; and European Patent Application 111,814). These documents relate to the insertion or deletion of bases at the 5' end of the BSt gene creating a protein different from the naturally-occurring polypeptide or, to changes in the BSt CDNA to maximize preferred codons and to reduce secondary structure in the mRNA, or to the use of a runaway plasmid to enhance expression. On the other hand, the instant invention teaches the use of an A T-rich ribosome binding site to produce heterologous polypeptides, and in particular, BSt at a high level.
Methods of culturing and fermenting transformed microorganisms expressing BSt are also referred to in the above-cited documents.
Purification of biologically active rBSt from transformed cells has also been described previously (see, e.g., U.S. Pat. Nos. 4,511,502, 4,511,503, 4,512,922 and 4,518,526; European Patent Application 131 843; and, Schoner, R. G., et al. , "Isolation and Purification of Protein Granules from E. coli Cells Overproducing BSt," Bio-Tech., 3:151-154 (1985)).